Fungal endophytes infect a number of temperate climate grass species. Endophytes can produce alkaloids which are considered to confer degrees of pest and possibly disease protection upon the plants in which they naturally occur (Rowan and Latch, 1994). Furthermore, the presence of at least some endophytes may be essential for the competitive persistence of the chosen grass in a pasture (Fletcher and Easton, 2000).
However, many of the predominating natural endophyte infections of improved grass cultivars used for pastoral agriculture production also cause significant disorders in animals grazing on such grasses. Examples of such disorders include tall fescue toxicoses (Stuedemann and Hoveland, 1988) and ryegrass staggers (Fletcher et al., 1999). These conditions are the result of complex toxic reactions by animals to alkaloids produced under a range of plant growth conditions. Significant economic loss within pastoral agriculture systems can occur due to such animal toxicoses.
Grass lines can be artificially infected with selected beneficial endophytes that only produce desirable alkaloids. Axenic cultures of endophytes can be used to infect grass seedlings, grown initially under sterile conditions, which are then selected for desirable qualities, and multiplied for commercial use.
Three significant examples of this technology have been developed by the Grasslands division of AgResearch Ltd: GREENSTONE™ tetraploid hybrid ryegrass with ENDOSAFE™ endophyte (NZ Patent 233083); various perennial and hybrid ryegrasses with AR1 endophyte (Fletcher and Easton, 2000); and tall fescue cultivars with MaxQ® endophyte (U.S. Pat. No. 6,111,170).
When preparing seeds with beneficial endophyte strains for commercial production, it is possible for seed batches to contain other contaminating and potentially harmful endophyte strains. It is thus important to have means available to test the endophyte content (beneficial and/or non-beneficial) of such seeds batches and/or parental material. In addition it is important to know if any endophyte present is viable, that is able to grow with the plant to form a stable symbiosis.
Various methods have been developed to test plant tissues, and seed, for the presence of endophytes.
Some methods such as microscopy, with histological staining with compounds such as aniline blue, only detect the presence of endophytes, and do not identify easily or reliably identify the species or strain of endophyte. In addition, current approaches using microscopy do not distinguish between viable and non-viable endophytes.
Monoclonal antibody kits can be used to indicate at a gross level the type of endophyte present by determining what alkaloids are being produced. But such kits cannot determine the actual species or strain.
High Performance Liquid Chromatography (HPLC) can be used to quantify toxic alkaloids produced by endophytes, which can be correlated with endophyte strains present. However again, this method does not distinguish between viable and non-viable endophytes.
Nucleic acid methods, such as polymerase chain reaction (PCR), have been used to detect the presence of specific strains in grass seed and plant tissue, but current approaches lack sensitivity and require significant time to grow plants to a suitable stage of development before they can be tested for the presence of endophyte. Furthermore, current methods do not distinguish viable from non viable endophyte.
Isozymes can be used to profile endophytes but again this method does not distinguish between viable and non-viable endophytes.
The current industry standard method for detecting the presence of viable endophytes requires live endophyte to grow out of host plant tissue onto axenic media to confirm viability. Enzyme Linked Immunosorbant Assay (ELISA) is then typically used to determine the percentage of contamination based on alkaloid profile, and the Western blotting (protein immunoblot) analytical technique is used to determine the presence or absence of endophyte. This method is labour intensive and time consuming. Furthermore, such serological methods often cannot distinguish between endophyte strains that produce similar alkaloid profiles.
Thus, many of the currently available methods are time consuming and expensive. Some of the methods also lack sensitivity, and/or are not amenable to screening of multiple samples, with a quick turnaround time from seed to result. Furthermore, as discussed, many of such methods are unable to distinguish between the presence of viable and non-viable endophytes.
It is an object of the invention to provide a method for detecting the presence of viable endophyte in plants that overcomes one or more of the difficulties associated with the prior art, and/or at least to provide the public with a useful choice.